a) Field of the Invention
This invention relates to the use of ligands specific to a Major Histocompatibility Complex (MHC)- class I antigen, especially an HLA-ABC surface antigen, which is normally exposed at the surface of cell membranes, and which is therefore present in or on endometrial cell, for the diagnosis of endometriosis. The detection of this antigen is carried out according to a process comprising the reaction of a ligand, preferably an antibody, which is normally used to detect the presence of a MHC-class I antigen at the surface of all cells expressing it.
This invention also relates to a method for the diagnosis of endometriosis using the same ligand(s).
b) Description of Prior Art
Endometriosis is one of the most common disorders encountered in the field of gynaecology, affecting the health of an estimated 10 to 15% of women during their reproductive years. Although not life threatening, endometriosis is often associated with severe pelvic pain and infertility.
Endometriosis is classically defined as the presence of endometrial tissue (i.e. glands and stroma) outside the uterine cavity which is its normal location. Although various hypothesis have been proposed for its pathogenesis (i.e. transplantation, lymphatic and vascular metastasis, or coelomic metaplasia), there are several lines of evidence to support the idea that retrograde menstruation and implantation are the primary mode of developing pelvic endometriosis.
The implantation theory proposes that viable endometrial tissue is refluxed through the fallopian tubes and implants on the peritoneal surface or pelvic organs. Some additional etiologic factors must be present for the development of endometriosis and could be implicated in an altered immune function.
To date, there are no sensitive reliable, non-invasive methods for the diagnosis of endometriosis.
Over the last decade, an increasing number of reports suggest that endometriosis is associated with abnormal immune function. It was first suggested, in 1980, that changes in humoral immunity cause endometriosis. The C3 components of the complement and IgG antibodies have been found in the endometrium of women with endometriosis, with a reduction in the levels of total complement. In addition, IgG and IgA antibodies have been already identified in the ovarian and endometrial tissue of women with endometriosis.
Recently, a high incidence of autoantibodies to phospholipids (particularly phosphatidylserine) and to histones and nucleotides has also been reported (Gleicher, 1987), suggesting polyclonal B-cell activation in endometriosis. The presence of more generalized autoantibodies have suggested that endometriosis could be an autoimmune disease. The evidence for autoimmune involvement is, however, far from unequivocal and many important questions remain.
The possibility that cell-mediated immunity is altered in women with endometriosis was first suggested in 1980. The concentration and total number of peritoneal macrophages are increased in endometriosis as well as their activator status. The contribution of reactive oxygen metabolites (superoxide anion (O2xe2x88x92), hydrogen peroxide (H2O2) and singlet oxygen (1O2)) to the damage of adjacent normal tissues in the presence of endometriosis has been suggested by an increased chemiluminescence of peritoneal macrophages.
More recently, a defect in natural killer cell (NK) activity has been reported. This reduced activity of the NK cells could also be present locally, at the level of the peritoneal fluid. Moreover, women with endometriosis show significantly more T-cell suppressor/cytotoxic in their peritoneal fluid.
Even though a defect in the activity of the NK cells has been noted in the presence of endometriosis, an increased resistance of the endometrial cells to the NK mediated cytotoxicity was also suggested. The mechanisms involved in this endometrial resistance have remained unexplained.
In the last five years, a considerable amount of research has been directed towards the development of a better diagnostic method for endometriosis. However, a problem lies in the fact that women suffering from endometriosis may not exhibit any symptoms other than infertility or they may only exhibit symptoms that mimic those of many other gynaecological diseases.
Hitherto, it has been common to use an invasive surgical method requiring general anesthesia to diagnose endometriosis, such as laparoscopy or laparotomy which allows the direct visualization of the pelvic content. However, there are certain disadvantages in using this difficult and inaccurate method for diagnosing endometriosis.
One such disadvantage of this method is that it is sometimes difficult to detect cases of minimal endometriosis. In addition, other disadvantages exist resulting from, for example, the presence of adhesions which can obscure the direct visualization of the pelvis, making it impossible to even carry out a laparoscopy; or from the presence of ovarian endometriomas which are often similar in appearance to functional ovarian cysts and could result in these endometriomas being misdiagnosed. Moreover, up to sixteen descriptive types of endometriosis have been identified recently, which can make the diagnosis of endometriosis even more difficult. Furthermore, what is even more worrisome is the fact that certain microscopic foci of endometriosis, which are not identifiable by laparoscopy, have been identified and documented using a peritoneal biopsy by scanning with an electron microscope.
Therefore, even with an increased use of laparoscopy, endometriosis remains a frequently underdiagnosed condition.
In general, imaging techniques such as ultrasonography, CT scanning, and magnetic resonance imaging (MRI) have limited value in the diagnosis of endometriosis. They can provide information about the invasiveness of the disease, but none of these imaging techniques are able to identify superficial diseases of peritoneal surfaces. Moreover, the high cost of these procedures rarely justifies their use for the diagnosis of endometriosis.
The technique of using antibodies like, for instance, anti-endometrial antibodies as mentioned hereinabove in a method for the diagnosis of various diseases is a widespread and well known technique.
In U.S. Pat. No. 4,444,744, an improved method is provided for using radiolabelled antibodies to detect the presence of cell surface antigens, including the type HLA-A, HLA-B AND HLA-DR surface antigens located on cancer cells, in order to locate and diagnose these cancer cells. These highly specific radiolabelled antibodies against the cell surface antigens are also used in a method for tumour therapy.
In U.S. Pat. No. 4,666,845, labelled mouse monoclonal antibodies, namely MF116, MH94, MD144, MH55, MF61, ME46, and ME195 were used to detect the presence of a plurality of surface antigen on human endometrial, cervical or uterine cancer cells and to diagnose these cancer cells.
These labelled monoclonal antibodies are also used in the treatment of endometrial, cervical or uterine cancers.
In Canadian patent publication number 2,081,900, a method of diagnosing endometriosis is described. This method detects the presence of particular antibodies which are present in specimens obtained from patients suffering of endometriosis. The antigens used as an immunological reagent were isolated from the cytoplasm of epithelial adenocarcinoma cells. These antigens have been characterized only by their molecular weight and by their ability to bind the antibodies to be detected.
In Canadian patent publication number 2,011,704, two methods of diagnosing endometriosis are described. One makes use of an antibody, particularly the MS2B6 monoclonal antibody, to detect endometrial antigens, while the other makes use of antigens also isolated from epithelial carcinoma cells to detect the presence of anti-endometrial antibodies. No attempt is made in this application to clarify the similarity or the complete identity of the antigens of both methods neither to clarify the similarity or equivalence of the MS2B6 antibody with regard to the endometrial antibodies. Furthermore, the antigens isolated from carcinoma cells are also grossly characterized by their molecular weight and their ability to bind the antibodies to be detected.
The first two patents are directed to the use of monoclonal antibodies to detect surface antigens present on various cancer cells, including endometrial cancer cells, and to the diagnosis of these cancer cells. In all cases, the expression of surface antigens of cancer cells cannot be considered as being similar to surface antigens found on endometrial cells of a woman suffering from endometriosis.
Therefore, the particular techniques illustrated in these patent documents could not be used as a method for diagnosing endometriosis or for detecting the different expression of surface antigens on endometrial cells of a woman with endometriosis.
Semino et al (Fertility and Sterility (1995) 64(5): 909-916) described that major histocompatibility complex class I molecules may have a role in the genetic control of endometriosis. The tested endometrial cells were taken from healthy women (control) or form endometriotic foci (by laparoscopy). These authors suggest that endometrial cells expressing a high number of MHC class I molecules on their surface give a protective signal to autologous lymphoid effector cells. On a very limited panel of five patients, they observed that a variable number of HLA class I molecules was expressed by endometriotic cells (ranging from a reduction of 15% to an enhancement of 30%, when compared to endometrial cells collected contemporaneously in the uterine mucosa).
Even though this reference may suggest a role for MHC class I molecules in endometriosis, it does not teach any correlation between endometriosis and the number of cells expressing MHC class I molecules. It does not further teach a non-invasive method e.g. a diagnostic method wherein laparoscopy would be avoided, in other words, performed on any endometrial cell sample.
In light of these results, there is clearly a great need for a non-invasive diagnostic test which is more reliable than the previous techniques and which is easier for both the patient and the physician. This new test may decrease the need for laparoscopy in women with pelvic pain or infertility.
The Applicant has found surprisingly that a woman with endometriosis has a different expression of the Major Histocompatibility Complex (MHC) class I antigens (especially HLA-ABC surface antigens) in or on their endometrial cells, when compared with the expression of the same antigens of endometrial cells of a healthy woman. Possibly, this difference in the expression of the MHC class I antigens prevents their recognition and cytolysis by the NK (natural killer) cells. This therefore may explain why the endometrial cells found in the peritoneal cavity are not destroyed, following retrograde menstruation and their subsequent peritoneal implantation. The presence of a different expression of these antigens on endometrial cells is therefore predictive of endometriosis.
A large number of ligands,. particularly monoclonal antibodies, that bind the aforesaid antigens normally expressed on the surface of a large number of human cells are available. Some authors (Johnson et al., 1984 and Sakaguchi et al., 1985) have evaluated the antigenic expression of the HLA-ABC antigens on the surface of endometrial cells. However the expression of the HLA-ABC antigens has never been correlated with the presence or absence of endometriosis. This is the first proposal suggesting that endometrial cells from patients with endometriosis express MHC-class I antigens, especially the HLA-ABC antigens, differently from normal individuals or other patients.
Therefore, it is an object of the present invention to provide the clinicians with a new application for ligands specific to MHC-class I antigens, especially HLA-ABC antigens, this new application residing in the detection and diagnosis of endometriosis.
It is also an object of the present invention to provide a test for diagnosing endometriosis, especially a non-invasive and reliable screening test, using a ligand to these antigens for the diagnosis of endometriosis. This antigen is present in or on endometrial cells or cells having the same embryonic origin (especially endocervical cells). Therefore, this method can be practised on a specimen obtained from the endometrium of a patient and does not require a specimen sampled directly from the endometriotic foci. The detection of these antigens carried out according to a process comprising the reaction of a ligand, e.g. antibodies with the antigens normally expressed at the surface of endometrial cells.
According to a preferred embodiment of the invention, the test uses a ligand to an antigen originating from endometrial cells. The MHC-class I antigens to which it will be referred hereinbelow are defined as comprising HLA-ABC antigens and related molecules. Such related molecules include proteic precursors like immature proteins, portions of these antigens as well as messenger RNAs and cDNAs. When proteins are targeted, the ligand of choice is an antibody specific thereto, and when messenger RNAs encoding these proteins are targeted, nucleotidic probes hybridizable thereto might be used to detect the difference of expression of these antigens.
MHC class I antigens, especially HLA-A, B and C antigens, are human cell surface glycoproteins. It is generally assumed that the Major human Histocompatibility Complex class I antigens, are expressed by nucleated cells with some exceptions like thymus, kidney or thyroid cells. They have a role in the interaction between cytotoxic T-cells and target cells.